A MULTIPLEX REAL-TIME PCR ASSAY FOR THE DETECTION AND TYPING OF Marteilia refringens

Arzul Isabelle*, Serpin Delphine, Maingourd Cyril, Boulet Hélène, Keck Nicolas, Stagg Hannah, Munro Eann, Civettini Michele, Arcangeli Giuseppe, Cheslett Deborah,  Savage Paul, Dubreuil Christine, Garcia Céline, Canier Lydie
 
 Laboratoire Génétique et Pathologie des Mollusques Marins
 Ifremer
Avenue de Mus de Loup
17390 La Tremblade
France
Isabelle.Arzul@ifremer.fr

Infection with Marteilia refringens is a notifiable mollusc disease to the EU and to the OIE. The host range of the parasite includes the flat oyster Ostrea edulis and mussels Mytilus edulis and M. galloprovincialis. Two types O and M previously considered as two distinct species, Marteilia refringens and M. maurini respectively have been characterized. Type O seems to occur more often in flat oysters while type M is more often detected in mussels although this host specificity is far from being strict. When M. refringens is detected by conventional PCR or in situ hybridization, complementary analyses are often carried out in order to specify parasite type.

In this context, we have recently developed a multiplex Taqman assay in order to detect and type M. refringens. By using one primer pair and two probes, this tool allows the concurrent detection and type characterization of the parasite.

Analytical specificity was checked against congeneric species and analytical sensitivity was evaluated using dilutions of plasmidic DNA suspensions for both M. refringens type M and type O.

As part of the validation process, an Inter Laboratory Comparison was organized to test the performance of the Taqman assay in seven laboratories. The test included 24 suspensions of ethanol fixed digestive gland tissue prepared from flat oysters, Ostrea edulis and mussels Mytilus edulis and M. galloprovincialis selected based on previous analyses and historical data.

The comparison study revealed a good relative sensitivity and specificity as well as a high repeatability and reproducibility (above 90%) of the real time PCR assay for the detection of both Marteilia refringens types.